Petr Svoboda

Impaired noradrenaline-induced lipolysis in white fat of aP2-Ucp1 transgenic mice is associated with changes in G-protein levels

In vitro experiments suggest that stimulation of lipolysis by catecholamines in adipocytes depends on the energy status of these cells. We tested whether mitochondrial uncoupling proteins (UCPs) that control the efficiency of ATP production could affect lipolysis and noradrenaline signalling in white fat in vivo. The lipolytic effect of noradrenaline was lowered by ectopic UCP1 in white adipocytes of aP2-Ucp1 transgenic mice, overexpressing the UCP1 gene from the aP2 gene promoter, reflecting the magnitude of UCP1 expression, the impaired stimulation of cAMP levels by noradrenaline and the reduction of the ATP/ADP ratio in different fat depots. Thus only subcutaneous but not epididymal fat was affected. UCP1 also down-regulated the expression of hormone-sensitive lipase and lowered its activity, and altered the expression of trimeric G-proteins in adipocytes. The adipose tissue content of the stimulatory G-protein alpha subunit was increased while that of the inhibitory G-protein alpha subunits decreased in response to UCP1 expression. Our results support the idea that the energy status of cells, and the ATP/ADP ratio in particular, modulates the lipolytic effects of noradrenaline in adipose tissue in vivo. They also demonstrate changes at the G-protein level that tend to overcome the reduction of lipolysis when ATP level in adipocytes is low. Therefore, respiratory uncoupling may exert a broad effect on hormonal signalling in adipocytes.

Molecular modeling of human MT2 melatonin receptor: the role of Val204, Leu272 and Tyr298 in ligand binding.

A model of the helical part of the human MT2 melatonin (hMT2) receptor, a member of the G protein‐coupled receptors superfamily has been generated, based on the structure of bovine rhodopsin. Modeling has been combined with site‐directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) numbers V, VI and VII of hMT2 receptor in the interaction with 2‐iodomelatonin. Saturation binding assays with 2‐iodomelatonin demonstrated that the substitution V204A (TMV) resulted in total loss of binding while the mutation V205A had no effect. The replacement of F209 with alanine led to a significant decrease in the Bmax value of receptor binding while mutations V205A and F209A also within TM V did not significantly change binding properties of the hMT2 receptor. In the case of TM VI, the substitution G271T caused substantial decrease in 2‐iodomelatonin binding to the hMT2 receptor. The change L272A (TM VI) as well as mutation Y298A within TM VII completely abolished ligand binding to the receptor. These data suggest that several new amino acid residues within TM V, VI and VII are involved in ligand–MT2 receptor interaction.

Opposing changes of trimeric G protein levels during ontogenetic development of rat brain.

Developmental changes in the distribution of guanine nucleotide-binding regulatory proteins (G proteins) were investigated in the rat brain during postnatal development. Using a standard or high-resolution urea-SDS-PAGE and specific polyclonal antipeptide antibodies oriented against G(i)alpha1/G(i)alpha2, G(i)alpha3, G(s)alpha, G(o)alpha1/G(o)alpha2, G(q)alpha/G(11)alpha and Gbeta subunit, all these proteins were determined by quantitative immunoblotting in homogenates prepared from cortex, thalamus, hippocampus and pituitary of 1-, 7-, 12-, 18-, 25- and 90-day-old animals. The levels of the majority of G protein alpha subunits, namely G(i)alpha1, G(i)alpha2, G(i)alpha3, G(o)alpha1, G(o)alpha2, G(q)alpha, G(11)alpha and Gbeta, were high already at birth. Whereas the short variant of G(s)alpha, G(s)alphaS, rose sharply in all tested brain regions between postnatal day (PD) 1 and 90, the long variant of G(s)alpha, G(s)alphaL, was unchanged in cortex and thalamus and slightly increased in hippocampus. An increase was observed also in expression of G(i)alpha1/G(i)alpha2 and G(o)alpha1 protein, while G(o)alpha2 remained constant. Minority protein G(o)alpha* dramatically increased in cortex and thalamus, was unchanged in hippocampus and not detectable in pituitary. By contrast, the highest levels of G(i)alpha3 and G(q)alpha/G(11)alpha were detected as early as at PD 1. During the next 90 days, the immunological signal of G(i)alpha3 almost disappeared and G(q)alpha/G(11)alpha continuously declined to the levels corresponding to 50% of the levels determined at birth. Expression of Gbeta subunit was basically unchanged during postnatal development. Our present analysis indicates that G(s)alpha, G(i)alpha/G(o)alpha and G(q)alpha/G(11)alpha proteins are differently expressed in the course of brain development. Differential expression of the individual alpha subunits of trimeric G proteins during postnatal development suggests their different roles in maturation of the brain tissue.

Ontogenetic development of the G protein-mediated adenylyl cyclase signalling in rat brain.

Maturation of the brain adenylyl cyclase (AC) signalling system was investigated in the developing rat cortex, thalamus and hippocampus. Expression of AC type II, IV and VI measured by Western blot dramatically increased in all tested brain regions during the first 3 weeks after birth and these levels were maintained in adulthood. AC type I did not change during ontogenesis. In parallel, AC enzyme activities were determined in order to obtain the functional correlates to the preceding structural (immunoblot) analyses of trimeric G proteins [Ihnatovych et al., Dev. Brain Res. (2002) in press]. Surprisingly, basal, manganese-, fluoride-, forskolin- and GTP-stimulated adenylyl cyclase developed similarly. The relatively low enzyme activities, which were determined at birth, progressively increased (about four times) to a clear maximum around postnatal day PD 12. This was followed by a progressive regression to adulthood so that activity of AC at PD 90 was comparable with the low neonatal level. The peak of AC activities at PD 12 was detected in all tested brain regions. Stimulatory (isoproterenol) effect on basal AC activity as well as inhibitory (baclofen) effect on forskolin-stimulated AC activity were unchanged between PD 12 and PD 90. Thus, comparison of results of the structural and functional analyses of adenylyl cyclase signalling system revealed a clear dissociation between the increase in the amount protein of various AC isoforms and the decrease of total G-protein mediated enzyme activities between PD 12 and adulthood. As none of the complex changes in trimeric G protein levels can explain this difference, the future research has to be oriented to identification of potential negative regulators of AC in the course of brain development. Among these, the newly discovered group of GTPase activating proteins, RGS, appears to be of primary importance because these proteins represent potent negative regulators of any G protein-mediated signalling in brain.

Thyrotropin-releasing hormone-induced depletion of Gqα/G11α proteins from detergent-insensitive membrane domains

The role of detergent‐insensitive membrane domains (DIMs) in desensitisation of the G protein‐coupled receptor‐mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin‐releasing hormone (TRH) receptors and G11α G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol‐bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the α subunit of Na,K‐ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin‐containing DIMs represented only a small fraction of the overall pool of Gqα/G11α‐rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of Gqα/G11α from all DIMs, which was paralleled by a concomitant Gqα/G11α increase in the high‐density gradient fractions containing the bulk‐phase membrane constituents soluble in 1% Triton X‐100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action.

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: correlation with studies of DPH and Laurdan fluorescence.

The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to wobble-in-cone model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of Gsα protein: identification of cytosolic forms of Gsα

Distribution of the α subunit of the stimulatory Gu2003protein (Gsα) was analyzed in membrane and cytosolic (supernatant 200u2003000u2003g) fractions from rat cortex, thalamus and hippocampus during the course of post‐natal development. In parallel, changes in β‐adrenoceptor density and adenylyl cyclase activity were determined. Long (GsαL) and short (GsαS) variants of Gsα were assessed by immunoblotting using specific polyclonal antisera reacting with both Gsα isoforms. Post‐natal development was associated with an increase in the total amount of brain Gsα. GsαL was the dominant isoform of Gsα in the membrane fractions of all studied brain regions and its amount increased markedly between post‐natal day (PD) 1 and 90. The level of membrane‐bound GsαS also elevated during post‐natal development, but more pronounced changes were found in cytosolic GsαS. Although only a small amount of GsαS (much smaller than GsαL) was detected among soluble proteins shortly after birth, GsαS prevailed over GsαL at PD90. The GsαL/GsαS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the GsαL/GsαS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane–cytosol balance of Gsα proteins alters dramatically during the course of brain development. Both GsαL and GsαS were expressed in a region‐ and age‐specific manner, which suggests different roles in the maturation of the brain tissue. A cyc− reconstitutive assay of cytosolic Gsα indicated that only ≈u200a20% of this protein was functional, compared with membrane‐bound Gsα, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of β‐adrenoceptors increased sharply during early post‐natal development but only slightly in adulthood, and both GTP‐ and isoproterenol‐stimulated adenylate cyclase activity reached peak values around PD12.

Membrane‐bound and cytosolic forms of heterotrimeric G proteins in young and adult rat myocardium: Influence of neonatal hypo‐ and hyperthyroidism

Membrane and cytosolic fractions prepared from ventricular myocardium of young (21‐day‐old) hypo‐ or hyperthyroid rats and adult (84‐day‐old) previously hypo‐ or hyperthyroid rats were analyzed by immunoblotting with specific anti‐G‐protein antibodies for the relative content of Gsα, Giα/Goα, Gqα/G11α, and Gβ. All tested G protein subunits were present not only in myocardial membranes but were at least partially distributed in the cytosol, except for Goα2, and G11α. Cytosolic forms of the individual G proteins represented about 5–60% of total cellular amounts of these proteins. The long (Gsα‐L) isoform of Gsα prevailed over the short (Gsα‐S) isoform in both crude myocardial membranes and cytosol. The Gsα‐L/Gsα‐S ratio in membranes as well as in cytosol increased during maturation due to a substantial increase in Gsα‐L. Interestingly, whereas the amount of membrane‐bound Giα/Goα and Gqα/G11α proteins tend to lower during postnatal development, cytosolic forms of these G proteins mostly rise. Neonatal hypothyroidism reduced the amount of myocardial Gsα and increased that of Giα/Goα proteins. By contrast, neonatal hyperthyroidism increased expression of Gsα and decreased that of Giα and G11α in young myocardium. Changes in G protein content induced by neonatal hypo‐ and hyperthyroidism in young rat myocardium were restored in adulthood. Alterations in the membrane‐cytosol balance of G protein subunits associated with maturation or induced by altered thyroid status indicate physiological importance of cytosolic forms of these proteins in the rat myocardium. J. Cell. Biochem. 82: 215–224, 2001.

FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: plasma membrane change induced by cholesterol depletion.

HEK293 cells stably expressing δ-opioid receptor were labeled first with fluorescent analog of cholesterol, 22-NBD-cholesterol, exposed to cholesterol-depleting agent β-cyclodextrin (β-CDX) and analyzed by fluorescence lifetime imaging microscopy (FLIM). In accordance with chemical analysis of cholesterol level, the total cellular signal of this probe was decreased to half. Distribution of lifetime (τtot) values of 22-NBD-cholesterol, however, when screened over the whole cell area indicated no significant difference between control (τtot=4.9±0.1 ns) and β-CDX-treated (τtot=4.8±0.1 ns) cells. On the contrary, comparison of control (τtot=5.1±0.1 ns) and β-CDX-treated (τtot=4.4±0.1 ns) cells by analysis of 25-NBD-cholesterol fluorescence implied highly significant decrease of lifetime values of this probe. The observation that 22-NBD-cholesterol appears to be indifferent to the changes in the membrane packing in living cells is in agreement with previous studies in model membranes. However, our data indicate that the alternation of plasma membrane structure induced by decrease of cholesterol level by β-CDX makes the membrane environment of NBD moiety of 25-NBD-cholesterol probe a significantly more hydrated. This finding not only encourages using 25-NBD-cholesterol in living cells, but also demonstrates that previously drawn discouraging conclusions on the use of 25-NBD-cholesterol in model membranes are not valid for living cells.

Differentiation of cultured brown adipocytes is associated with a selective increase in the short variant of Gsα protein. Evidence for higher functional activity of GsαS

Abstract In order to examine whether the differentiation process in brown adipocytes cultivated in primary culture is associated with substantial alterations in the complement of G proteins, the levels of these proteins were investigated with immuno-electrophoretic techniques in membrane preparations from proliferating and differentiated cultured mouse brown adipocytes. We observed that differentiation was associated with a dramatic (more than threefold) increase in the short variant of Gsα protein (GsαS). The long variant of Gsα (GsαL), as well as Gi1α, Gi2α, Gqα, G11α and Gβ subunit proteins remained unchanged whereas Gi3α protein was decreased. These changes were accompanied by marked increase in isoprenaline-, forskolin- as well as manganese-stimulated adenylyl cyclase. Thus, the marked increase in β-adrenergic responsiveness of fully differentiated confluent brown adipocytes (day 8–9), as compared with that of proliferating undifferentiated cells of ‘fibroblast phenotype’ (day 3–4), is associated with a significant increase in the relative proportion between the short and long variants of Gsα (the GsαS/GsαL ratio) along with a decrease in Gi3α protein. These data also suggest that the short variant of Gsα exhibits higher functional activity than the long variant of this G protein.